This invention relates to confirmatory assays and, in particular, to a novel confirmatory assay in which true positives are distinguished from false positives caused by related ligands or cross-reactive organisms, as well as false positives caused by non-specific assay artifacts.
Confirmatory assays are well known in the art and generally involve repeating with some modifications an initial assay which has detected the presence of a ligand, antigen or organism in a test sample. Known confirmatory assays use confirmatory binding proteins (CBPs), usually antibodies, which have specificities identical to the detector binding protein (DBP) of the detecting assay but which are unable to generate a signal. By addition of the CBP prior to the DBP, the binding sites normally occupied by the DBP are blocked and the signal is correspondingly reduced. For example, a commercially available (from Abbott Laboratories, Abbott Park, IL.) assay for confirming the presence of Hepatitis B Surface Antigen (HB.sub.s Ag) utilizes human anti HB.sub.s Ag as a CBP to block available sites on antigen in the sample. Then the assay is repeated, and a signal reduction of 50% confirms the presence of HB.sub.s Ag.
These assays eliminate extraneous signal generation caused by non-specific binding of the DBP to various assay components to give false positive signals. Such false positive signals caused by DBP binding to (i.e. detection of) assay components are referred to herein as "assay artifact" signals
In contrast, false positive signals may also be generated by DBP binding to ligands or organisms which closely resemble the target ligand or organism, or are cross-reactive therewith. Due to identical specificity between the DBP and the CBP, conventional confirmatory assays can eliminate assay artifact false positives, but not false positives originating from closely related or cross reactive ligands or organisms in the test sample. This disadvantage is overcome by the present invention.